Yersinia pestis is a Gram-negative bacillus, which is the etiological agent of plague. Human infection by this pathogen may occur by subcutaneous (e.g., flea bite), intranasal, or intravenous routes, resulting in bubonic or pneumonic plague or septicemia, respectively. The intentional dissemination of Y. pestis as a bioterroist act would likely occur as an aerosol causing pneumonic plague. This is the most severe form, with rapid onset of symptoms and high mortality if not treated appropriately. Novel agents not susceptible to existing resistance mechanisms and capable of facilitating theraputic intervention are needed for biodefense. This bacterium employs multiple non-redundant virulence factors to evade the innate immune system and the ensuing proinflammatory response in order to grow rapidly in the host and cause septicemic death. One of these virulence factors, a membrane-embedded surface plasminogen activator (Pla), a member of the omptin protease family, facilitates dissemination of Y. pestis cells in the infected host. Absence of Pla results in a million-fold reduction in virulence for subcutaneously infected mice (LD50 increases from 50 to 107 bacterial cells) and significantly reduces the rapidity of pneumonic disease progression. Furthermore, homologous omptins are virulence factors in E. coli, Salmonella and Shigella, enhancing evasion of the host innate immune system and facilitating inter- and intracellular movement. Therefore, broad spectrum inhibitors of this protease family will have multiple therapeutic applications, both clinically and for biodefense. The goal of this project is to identify specific inhibitors of Y. pestis Pla, select those with activity against multiple members of the omptin protease family, and develop them into novel agents for co-therapy against Y. pestis, E. coli, Salmonella, and/or Shigella infections. The cell surface accessibility of the target and the well-established "druggability" of protease targets argue for the feasibility of this approach. In preliminary studies, reliable assays were developed for inhibitors of cell membrane-associated Pla. Recombinant E. coli strains producing active outer- membrane anchored Y. pestis Pla at 40% of the outer membrane protein were constructed. In Phase I, the assay will be optimized as a high throughput screen for inhibitors of Y. pestis Pla bound to recombinant E. coli cells and applied to a diverse library of = 300,000 discrete small molecule compounds and purified natural products. Secondary assays will be developed to validate the inhibitory activity of confirmed screening hits against physiological activities of several omptin proteases, including inhibition of the degradation of alpha-2- antiplasmin and antimicrobial peptides, as well as inhibition of invasion of cultured cells by Y. pestis. Finally, compounds will be prioritized by demonstrating that they exhibit little or no mammalian cell toxicity and that they enhance liver microabscess formation in mice infected with Y. pestis. In Phase II, analogs of the highest priority hits will be chemically optimized to develop lead compounds. The most promising lead compounds will be tested for efficacy and toxicity in animal models. PUBLIC HEALTH RELEVANCE: This research is aimed at discovering new drugs which reduce the virulence of several bacterial pathogens, broadening the therapeutic window of time during which antibiotics may be appropriately selected and administered. Features of the drug target make discovery of effective agents particularly feasible. These new drugs will not be susceptible to existing resistance mechanisms and will provide a proof of principle for the efficacy of this therapeutic approach.